Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene cluster
Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene cluster
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DOI：
10.1128/aem.62.10.3567-3571.1996
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发表时间：
1996-10-01
影响因子：
4.4
通讯作者：
Woloshuk, CP
中科院分区：
文献类型：
--
作者：
Prieto, R;Yousibova, GL;Woloshuk, CP
黄曲霉突变株 649 具有覆盖黄曲霉毒素生物合成簇的至少 120 kb 的基因组 DNA 缺失，用一系列重叠的粘粒进行转化，这些粘粒含有带有该基因簇的 DNA。菌株649的突变表型通过用粘粒克隆5E6、8B9和13B9的组合转化得以挽救，表明参与黄曲霉毒素生物合成的基因簇位于这些克隆携带的黄曲霉基因组DNA的90kb中，转化子5E6和20B11以及转化子5E6和8B9积累了黄曲霉毒素的中间代谢产物。黄曲霉毒素途径，分别被鉴定为averufanin和/或averufin，这些数据表明，avf1存在于粘粒13B9中，该avf1参与将averufin转化为versiconal半缩醛乙酸酯，13B9的删除分析将该基因定位在粘粒的7-kb DNA片段上，含有外源提供的粘粒8B9转化的转化体O-甲基杂色曲霉素转化为黄曲霉毒素，表明介导O-甲基杂色曲霉素转化为黄曲霉毒素的氧化还原酶基因(ord1)由该粘粒携带。对含有8B9缺失的转化子的分析导致ord1定位在粘粒的3.3-kb黄曲霉基因组DNA片段上。
Aspergillus flavus mutant strain 649, which has a genomic DNA deletion of at least 120 kb covering the aflatoxin biosynthesis cluster, was transformed with a series of overlapping cosmids that contained DNA harboring the cluster of genes. The mutant phenotype of strain 649 was rescued by transformation with a combination of cosmid clones 5E6, 8B9, and 13B9, indicating that the cluster of genes involved in aflatoxin biosynthesis resides in the 90 kb of A, flavus genomic DNA carried by these clones, Transformants 5E6 and 20B11 and transformants 5E6 and 8B9 accumulated intermediate metabolites of the aflatoxin pathway, which were identified as averufanin and/or averufin, respectively, These data suggest that avf1, which is involved in the conversion of averufin to versiconal hemiacetal acetate, was present in the cosmid 13B9, Deletion analysis of 13B9 located the gene on a 7-kb DNA fragment of the cosmid, Transformants containing cosmid 8B9 converted exogenously supplied O-methylsterigmatocystin to aflatoxin, indicating that the oxidoreductase gene (ord1), which mediates the conversion of O-methylsterigmatocystin to aflatoxin, is carried by this cosmid, The analysis of transformants containing deletions of 8B9 led to the localization of ord1 on a 3.3-kb A. flavus genomic DNA fragment of the cosmid.